Dyslipidemia is a major risk factor for premature atherosclerotic cardiovascular disease (ASCVD). Although many patients with dyslipidemia can be treated effectively with existing drugs, others are not effectively treated and remain at exceptionally high risk of premature ASCVD; the classic example is homozygous familial hypercholesterolemia (FH). Therefore, understanding of the regulation of the secretion and catabolism of apoB-containing lipoproteins by the liver and the complex pathways of HDL metabolism is of major importance to the development of new therapies targeted toward these pathways. Liver-directed somatic gene transfer is a useful biological tool for addressing hypotheses regarding the physiological effects of expressing specific genes in the liver on apoB lipoprotein and HDL metabolism; furthermore, it could be a strategy for treating severe dyslipidemia. In this project, we will utilize liver-directed gene transfer using vectors based on novel adeno-associated virus (AAV) pseudotypes (as described in Project 1) to address questions related to the impact of specific gene products and their interactions on the regulation of hepatic secretion of apoB-containing lipoproteins. These projects have been designed to leverage the substantial advantages of vectors based on novel AAV pseudotypes, such as AAV2/8, including the stable and efficient transduction of hepatocytes after intravenous administration without apparent inflammation. AAV2/8-based vectors are a major advance over AAV2-based vectors with regard to levels of expression (more than an order of magnitude in our experience) and over adenoviral based vectors because of the lack of inflammation (compared to severe inflammation with adenoviral vectors) and the stable long-term expression (compared to transient expression with adenoviral vectors). Specific Aim 1. To determine the roles of hepatic DGAT1 and DGAT2 expression in affecting hepatic VLDL TG and apoB production through AAV-mediated gene transfer of cDNAs to achieve overexpression through AAV-mediated gene transfer of RNAi to achieve reduction of expression. To test the hypothesis that hepatic MTP expression interacts with hepatic DGAT expression in influencing hepatic VLDL production. Specific Aim 2. To test the hypothesis that AAV-mediated overexpression of the LDL receptor (LDLR) or VLDL receptor (VLDLR) increases presecretory apoB degradation, reducing the hepatic VLDL triglyceride and apoB production rate. To determine whether AAV-mediated LDLR and VLDLR overexpression can normalize VLDL production in mouse models of VLDL triglyceride and apoB overproduction. Specific Aim 3. To compare AAV-mediated liver-directed gene transfer of the LDLR and the VLDLR in LDLR/apobec-1 double knockout mice on progression and regression of pre-existing atherosclerosis. To compare AAV-mediated gene transfer of the LDL receptor and the VLDL receptor in WHHL rabbits on lipoprotein metabolism and atherosclerosis. Specific Aim 4: To use AAV-mediated liver-directed gene transfer of apoA-I to test whether long-term expression of apoA-I can prevent progression and induce regression of atherosclerosis and in murine and rabbit models of atherosclerosis. To test whether long-term stable expression of apoA-I is additive to long-term cholesterol reduction with regard to atherosclerosis progression and regression. The overall goals of this grant proposal are to use AAV-mediated liver-directed gene transfer: 1) to test specific hypotheses related to the role of DGAT1 and DGAT2 in the regulation of hepatic VLDL TG and apoB production in vivo through gene transfer-mediated overexpression and gene knockdown approaches; 2) to compare gene transfer of the LDLR and the VLDLR with regard to effects on VLDL production, on plasma lipoproteins, and on atherosclerosis in two different animal models of FH; and 3) to generate proof-of-principle that AAV-mediated gene transfer of apoA-I to liver can induce regression and prevent progression of atherosclerosis in mice and rabbits. This project will generate new biological information and will also generate proof of principle in relevant animal models of homozygous familial hypercholesterolemia and low HDL cholesterol that would support the concept of gene transfer as a potential therapeutic approach to these disorders.